There are many instances whereby the efficacy of a therapeutic protein is limited by an unwanted immune reaction to the therapeutic protein. Several mouse monoclonal antibodies have shown promise as therapies in a number of human disease settings but in certain cases have failed due to the induction of significant degrees of a human anti-murine antibody (HAMA) response [Schroff et al. (1985) Cancer Res. 45: 879-885; Shawler et al. (1985) J. Immunol. 135: 1530-1535]. For monoclonal antibodies, a number of techniques have been developed in attempt to reduce the HAMA response [WOA8909622; EPA0239400; EPA0438310; WOA9106667; EPA0699755]. These recombinant DNA approaches have generally reduced the mouse genetic information in the final antibody construct whilst increasing the human genetic information in the final construct. Notwithstanding, the resultant “humanised” antibodies have, in several cases, still elicited an immune response in patients [Issacs J. D. (1990) Sem. Immunol. 2: 449, 456; Rebello et al. (1999) Transplantation 68: 1417-1420].
Antibodies are not the only class of polypeptide molecule administered as a therapeutic agent against which an immune response may be mounted. Even proteins of human origin and with the same amino acid sequences as occur within humans can still induce an immune response in humans. Notable examples include therapeutic use of granulocyte-macrophage colony stimulating factor [Wadhwa et al., (1999) Clin. Cancer Res. 5: 1353-1361] and interferon α2 [Russo et al. (1996) Bri. J. Haem. 94: 300-305; Stein et al. (1988) New Engl. J. Med. 318: 1409-1413] amongst others.
Key to the induction of an immune response is the presence within the protein of peptides that can stimulate the activity of T-cells via presentation on MHC class II molecules, so-called “T-cell epitopes.” Such T-cell epitopes are commonly defined as any amino acid residue sequence with the ability to bind to MHC Class II molecules. Implicitly, a “T-cell epitope” means an epitope which when bound to MHC molecules can be recognized by a T-cell receptor (TCR), and which can, at least in principle, cause the activation of these T-cells by engaging a TCR to promote a T-cell response.
MHC Class II molecules are a group of highly polymorphic proteins which play a central role in helper T-cell selection and activation. The human leukocyte antigen group DR (HLA-DR) are the predominant isotype of this group of proteins however, isotypes HLA-DQ and HLA-DP perform similar functions. In the human population, individuals bear two to four DR alleles, two DQ and two DP alleles. The structure of a number of DR molecules has been solved and these appear as an open-ended peptide binding groove with a number of hydrophobic pockets which engage hydrophobic residues (pocket residues) of the peptide [Brown et al., Nature (1993) 364: 33; Stern et al. (1994) Nature 368: 215]. Polymorphism identifying the different allotypes of class II molecule contributes to a wide diversity of different binding surfaces for peptides within the peptide binding grove and at the population level ensures maximal flexibility with regard to the ability to recognise foreign proteins and mount an immune response to pathogenic organisms.
An immune response to a therapeutic protein proceeds via the MHC class II peptide presentation pathway. Here exogenous proteins are engulfed and processed for presentation in association with MHC class II molecules of the DR, DQ or DP type. MHC Class II molecules are expressed by professional antigen presenting cells (APCs), such as macrophages and dendritic cells amongst others. Engagement of a MHC class II peptide complex by a cognate T-cell receptor on the surface of the T-cell, together with the cross-binding of certain other co-receptors such as the CD4 molecule, can induce an activated state within the T-cell. Activation leads to the release of cytokines further activating other lymphocytes such as B cells to produce antibodies or activating T killer cells as a full cellular immune response.
T-cell epitope identification is the first step to epitope elimination as recognised in WO98/52976 and WO00/34317. In these teachings, predicted T-cell epitopes are removed by the use of judicious amino acid substitutions within the protein of interest. Besides computational techniques, there are in vitro methods for measuring the ability of synthetic peptides to bind MHC class II molecules. An exemplary method uses B-cell lines of defined MHC allotype as a source of MHC class II binding surface and may be applied to MHC class II ligand identification [Marshall et al. (1994) J. Immunol. 152:4946-4956; O'Sullivan et al. (1990) J. Immunol. 145: 1799-1808; Robadey et al. (1997) J. Immunol 159: 3238-3246]. However, such techniques are not adapted for the screening multiple potential epitopes to a wide diversity of MHC allotypes, nor can they confirm the ability of a binding peptide to function as a T-cell epitope.
Recently, techniques exploiting soluble complexes of recombinant MHC molecules in combination with synthetic peptides have come into use [Kern et al. (1998) Nature Medicine 4:975-978; Kwok et al (2001) TRENDS in Immunol. 22:583-588]. These reagents and procedures are used to identify the presence of T-cell clones from peripheral blood samples from human or experimental animal subjects that are able to bind particular MHC-peptide complexes and are not adapted for the screening multiple potential epitopes to a wide diversity of MHC allotypes.
As depicted above and as consequence thereof, it would be desirable to identify and to remove or at least to reduce potential T-cell epitopes from a given in principal therapeutically valuable but originally immunogenic peptide, polypeptide or protein. One of these therapeutically valuable molecules is a monoclonal antibody with binding specificity for the CD52 human leukocyte antigen. The preferred monoclonal antibody of the present invention is a modified form of the rat antibody termed “CAMPATH”. It is an objective of the invention to provide for modified forms of the CAMPATH antibody with one or more potential T-cell epitopes removed.
The CD52 molecule has a molecule weight of 21-28 kDa, and the mature protein comprises a 12 amino acid peptide with a N-linked oligosaccharide being attached to the membrane by its glycophosphatidylinositol anchor. The antigen is present on at least 95% of human peripheral blood lymphocytes and also cells of the monocyte/macrophage series and in addition spermatozoa. It is not present on erythrocytes, platelets, tissue dendritic cells or bone marrow stem cells (Hale et al. (1990) Tissue Antigens 35:873; Buggins et al (2002) Blood, 100:1715).
The first CD52 antibodies were raised in a rat immunized with human lymphocytes in an attempt to obtain antibodies that activated complement for use to deplete donor marrow of T-cells prior to transplantation [Hale et al. (1983) Blood 62: 873-882]. The majority of lytic antibodies were anti-CD52 IgM antibodies. Although useful ex vivo, CD52 IgM (CAMPATH-1M) mediated complement activation was not effective in eliminating T-cells in vivo. CAMPATH-1G, a rat IgG2b monoclonal antibody, obtained by isotype switching from an IgG2a antibody clone, binds human Fc receptors, mediates cell death antibody-mediated cellular toxicity (ADCCD) and is effective in eliminating cells in vivo [Friend et al. (1991) Transplant. Proc. 23: 2253-2254; Hale et al. (1998) Blood 92: 4581-4590]. However, use of CAMPATH-IG is limited by the immune response elicited in patients [Cobbold, J. S. (1990) J. Immunol. Methods 127: 19-24; Dyer, M. J. S. (1989) Blood 73: 1431-1439]. To reduce immunogenicity, a humanized IgG1 antibody, CAMPATH-1H, was engineered by cloning the Kabat hypervariable regions into a framework provided from human NEW and Rei myeloma proteins [Riechmann et al., (1988) Nature 332: 323-327]. Although reducing the immunogenicity compared to CAMPTH-1G, the humanized antibody still elicits immune responses in some patients. In an early report of treatment for rheumatoid arthritis, no antiglobulin response was reported in the 8 patients treated by a first course of i.v. administration, but 3 of 4 patients who received a second course of CAMPATH-1H developed antiglobulin antibodies (Issacs et al. (1992) Lancet, 21:1103-06). In a subsequent single-dose escalation i.v. study in rheumatoid arthritis patients, 63% of subjects developed antiglobulin responses, which were primarily anti-idiotypic responses [Weinblatt et al. (1995) Arthritis. Rheum. 38: 1589-1594]. Antiglobulin responses were also reported for all 10 rheumatoid arthritis patients who received escalating doses of CAMPATH-1H by subcutaneous administration (Schnitzer et al., J. Rheumatol. (1997) 24:1031-36).
Thus, it is desirable to provide anti-CD52 antibodies with a reduced number of potential T-cell epitopes which may result in a reduced or absent potential to induce an immune response in the human subject. Such proteins may be expected to display an increased circulation time within a human subject capable of mounting an immune response to the non-modified antibody and may be of particular benefit in chronic or recurring disease settings such as is the case for a number of indications for CAMPATH. The present invention accordingly provides for modified forms of an anti-CD52 antibody with reduced numbers of potential T-cell epitopes that are expected to display decreased immunogenicity while however, substantially retaining the beneficial therapeutic features associated with the efficacy of the parental non-modified antibody.
The invention discloses sequences identified within the variable region sequences of the heavy and light chains of an anti-CD52 antibody that are potential T cell epitopes by virtue of MHC class II binding potential.
While others have provided anti-CD52 antibody molecules including “humanised” forms [U.S. Pat. Nos. 5,846,543; 6,120,766; 6,569,430; WO0230460] none of these teachings recognise the importance of T cell epitopes to the immunogenic properties of the protein nor have been conceived to directly influence said properties in a specific and controlled way according to the scheme of the present invention.